Transcription Factor Overexpression Drives Reliable Differentiation of Retinal Pigment Epithelium From Human Induced Pluripotent Stem Cells

We report a robust, scalable method to generate iPSC-RPE using doxycycline-inducible expression of eye field transcription factors OTX2, PAX6 and MITF paired with RPE-permissive culture media. Doxycycline addition induces exogenous expression of these transcription factors in patient- and wildtype iPSCs to efficiently produce monolayers of RPE with characteristic morphology and gene expression. Further, these RPE monolayers display functionality features including light absorption via pigmentation, polarity-driven fluid transport, and phagocytosis. With this method, we achieve a highly efficient and easily scalable differentiation without the need for mechanical isolation or enrichment methods, generating RPE cultures applicable for in vitro studies. We analyzed samples from 3 different stages of the differentiation for each cell line, with 3 replicates each, resulting in 18 samples total.